Notes on Genetics of Bacteria Describe the structure of bacterial chromosomes including the arrangement of DNA within bacterial cells

s o Bacteria are prokaryotes having very simple internal structure with no membrane bound organelles o The bacterial chromosomes are found in the nucleoid region (area where chromosome is found but not bounded by membrane) o Double stranded circular DNA molecule (eu chromosomes are linear) o Associated with several types of DNA binding proteins but much fewer than the eukaryotic chromosomes, thus considered naked Two called HU and H are small but abundant Contain high percentage of positively-charged amino acids that bind ionically to the negative charges of DNA Structurally similar to histones HU protein critical to maintenance of the nucleoid structure o Functionally active (not inert) o Replicate by binary fission o Readily replicated and transcribed. Transcription and translation usually take place at the same time in bacteria o Has negative supercoiling when DNA is twisted about its axis in the opposite direction from that of the right-handed double helix which is necessary for packing the DNA into the confines of a cell and relaxing is necessary so DNA can be replicated/transcribed o Many bacteria often contain one or more small circular non-chromosomal DNA molecules called plasmids Double-stranded, circular Autonomously replicating molecules that is separate from the bacteria cells chromosomes Code for synthesis of a few proteins not coded for by the nucleoid Not essential for normal bacteria growth but usually contain supplemental information that might confer some characteristics that can provide advantage under certain environmental conditions Fertility / mating capabilities Resistance to antibodies Ability to produce toxins Tolerance to toxic materials Degradative / metabolism of nutrients Also function as vectors in recombinant DNA technology. Contain genetic markers which confer well-defined phenotypes on the host cell. (ie have restriction sites that can just nice cut by restriction the exact portion of the DNA where it codes for that particular phenotype) Bacterial Genome Eukaryote genome - Haploid - Diploid o May contain more than 1 copy of its o Contain two complete copies of its genome during rapid growth genome o But these copies are identical o Not all genes on the two sets are identical result of meiosis - Little Redundancy of DNA - More Redundancy of DNA o Nearly entire genome is used for coding or o In humans >95% of genome is non-coding regulation o In E.coli, >70% used for coding - Not arranged in Nucleosomes - Arranged in nucleosomes 1|Page Created by SefLRho (2012) DHS

o Usually naked Few introns Clustering of genes encoding related functions o Function in operons system o Operon is controlled as one unit to produce a polycistronic messenger RNA (1 mRNA that transcribe several structural genes ie more than 1 protein coded in that 1 mRNA)

o Arranged by histones Lots of introns No-clustering o 1 mRNA code for 1 gene

Describe the process of binary fission in bacteria o Binary fission is a form of asexual reproduction that results in the reproduction of a living prokaryotic cell by division into two equal halves, each having the potential to grow to the size of the original cell o DNA replication DNA replication begins at a single origin of replication (ori) which opens up into replication bubble DNA synthesis progresses in both directions around the circular chromosome outwards from the origin of replication 2 copies of bacterial chromosome result o Cell elongation Cell grows in size and elongates o DNA segregation Two copies of bacterial chromosome attached to the mesosome The newly the original chromosomes move to opposite ends of the cells o Cell division When bacterium grows the cell membrane invaginates and splits the cell into two. This is controlled by septal ring (a collection of about a dozen proteins) Finally cell wall is deposited in the middle of the cell and two daughter cells are separated o Advantages of binary fission Bacteria can proliferate very rapidly Most bacteria in a colony are genetically identical to the parent cell because it is an asexual process Although mutations are rare in asexual reproduction, it still occurs and have significant impact on the genetic diversity since reproductive rates are high Other than mutations, genetic recombination generates diversity within bacterial populations Describe genetic recombination and the various methods of achieving variety (transformation, transduction, conjugations) o Genetic recombination refers to the combining of DNA from two individuals into the genome of a single individual o A cell which undergoes genetic recombination is called a recombinant. Its chromosomes contain DNA derived from two different cells Describe the process of transformation in bacteria o Transformation is the alteration of a bacterial cells genotype by the uptake of naked, foreign DNA from the surrounding environment The foreign DNA may come from dead, lysed neighbouring cells or deliberately introduced in laboratory conditions

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Requirements for transformation Cell competency cell that is able to take up exogenous DNA and be transformed natural competence: is regulated by special proteins (competencespecific protein) Artificial competence: introduced by heat-shock treatment Uptake of DNA can be stimulated by using high concentration of calcium ions in artificial settings Presence of naked DNA Double-stranded DNA binds more effectively to the cells Although only one strand ultimately enters the cell as the other is degraded during the uptake process Process of transformation Transforming DNA bind at DNA binding protein (protein receptor) After uptake, the DNA associates with competence-specific proteins that remain attached to the DNA to keep it from nuclease attack The DNA is integrated into the bacterial genome by homologous recombination mediated by RecA protein

Describe the process of transduction in bacteria o Transduction is the transfer of DNA from cell to cell through the agency of viruses

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Phages carry bacterial genes from one host cells to another as a result of aberrations in the phage reproductive cycle o Transducing viruses are usually defective as a virus because bacterial genes have replaced some necessary viral genes o Generalized transduction Occurs in the lytic cycle Phage infects the host by injecting its DNA Phage enzymes hydrolyse the bacteria chromosomes Occasionally, a small piece of the host cells degraded DNA is accidentally packaged within a phage capsid by mistake in place of the phage genome Such virus is defective because it lacks its own genetic material -> no new phage can be synthesized in the recipient cell The infected bacterial cells lyses and release the defective phage Phage attach to another bacterium and injects the piece of bacterial DNA along with its own DNA Results (either): Absorbed by cell and recycled for spare parts Re-circularize inside the new cell and becomes a plasmid again if the DNA was originally a plasmid Exchange DNA material by homologous recombination, if the new DNA matches with a homologous region of the recipient cell (crossing over) o Specialized transduction Requires infection by a temperate phage Occurs in the lysogenic cycle The genome of the a temperate phage integrates as a prophage at a specific site Later when the phage genome is excised from the chromosome, it sometimes takes with it a small region of the bacterial DNA that was adjacent to the prophage When such a virus infects another cell, the bacterial genes are injected along with the phages genome Results (either): DNA absorbed and recycled for spare parts Exchange DNA material by homologous recombination if the new DNA matches with ah homologous region of the recipient cells chromosomes (crossing over) Insert itself into the genome of the recipient cell as if still acting like a virus resulting in a double copy of the bacterial genes Describe the process of conjugations in bacteria o Conjugation is the direct transfer of genetic material through direct cell-to-cell contact (mating bridge) between bacterial cells that are temporarily joined o The DNA transfer is one-way. The one with the F-plasmid as the donor and the one without as the recipient o The donor is able to form sex pili to attach to the recipient o the sex pilus acts like a grappling hook, retracting and pulling the two cells together o the cell membranes fuse, forming a temporary cytoplasmic bridge between the two cells o F factor give the ability to form sex pili and donate DNA during conjugation Donor = F+ Recipient = Fo F factor can exist either as segment of DNA within the bacterial chromosomes or as a F plasmid

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F+ = have F factor. F- = lacking F factor F plasmid is replicated by rolling circle replication starting at the origin of transfer One copy is passed from the F+ to F Once the F- cell receives the F plasmid, it is converted to F+ cell In an F+ x F- mating, only the F plasmid is transferred Explain the role of F plasmids in bacterial cells o Plasmid is a small, circular, self-replicating DNA molecule separate from the bacterial chromosomes o F plasmids can undergo reversible incorporation into the cells chromosomes by genetic recombination (when incorporated it is called F factor, when not, it is called f plasmid) o a genetic element that can exist either as a plasmid or as part of the bacterial chromosomes is called an episome o plasmids have only a small number of genes (about 40 genes) and are generally beneficial they code for the ability for make sex pili and other things needed for conjugation o F plasmid facilitates genetic recombination may be advantageous in a changing environment Genetic recombination may occur in the case that the F factor (F plasmid that is in integrated within the genome) replicates without excising itself from the genome such that it becomes F plasmid. So as the F factor replicate, it has its own origin of replication and thus serve to be the leading end of the transferred DNA, replication not only its gene but also genes from the donor original chromosome Then the replicated DNA (F factor + host genome) enters the F- cell and the host genome can undergo homologous recombi with the F- cell and cause genetic recombination o F factor is inheritable and control the plasmids replication and the synthesis by the host cell of a filamentous appendage called the sex pilus PLASMIDS Lack protein coat Do not normally exist outside the cell VIRUSES Has protein coat Has two forms Extracellular = virion Intracellular virus/provirus Are parasites and usually harm their hosts

Generally beneficial Bacterial gene expression o Bacteria do not make all the proteins that they are capable of making all the time o Rather, they adapt to their environment and make only those gene products that are essential for them to survive in that particular environment o The reason for such is: Efficient use of energy Economical use of resources Responsiveness to environment

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Control of metabolic reactions o Most the regulation of gene expression is controlled at the transcription level this is because it is the stage where it is most efficient with minimal wastage of the cells resources o Metabolic control in prokaryotes Adjust the amount of certain enzymes being made by regulating gene express (at level of transcription) Adjust the activity of the enzymes present by inhibition/activation/inactivation/degradation Eg end product inhibition allow cell to adapt to short-term fluctuations in level of substance it need Distinguish between structural and regulatory genes o Structural gene is a region of DNA that codes for a protein or RNA molecule that forms part of a structure or has an enzymatic function o Regulatory gene codes for a specific protein product that regulates the expression of the structural gene o Regulatory gene codes for DNA binding proteins Activators Repressors o Activities of these activators and repressors are dependent on the allosteric binding of small effectors molecules known as inducers and co-repressors Distinguish between concept of repressible and inducible systems of gene regulation o A repressible system is a system of enzymes whose transcription is usually on but can be repressed when a co-repressor binds allosterically to a regulatory protein o Usually found in anabolic pathways o The enzymes synthesizing the specific product of this operon would not be synthesized if the product is present in the medium o Eg trp operon o An inducible system is a system of enzymes whose transcription is usually off but can be induced when an inducer binds allosterically to the regulatory protein o Usually found in catabolic pathways o The enzymes coded for by the operon would only be synthesized when it he needed o Eg lac operon Trp operon

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Example of repressible operon Uses only negative regulation/control Tryptophan operon is usually on but can be repressed When the intracellular level of trp rises, the enzyme synthesis is repressed Repressor protein is synthesized in the inactive form (tryptophan, the co-repressor needs to bind to it to make it active) o Trp (the end product which also serves as co-repressor) must accumulate to high amount before it can bind to trp repressor to make it active o This is called end product repression (note that this is NOT end product inhibition. Inhibition happens at the enzyme level, this occurs at gene level) o Details 1. The tryptophan repressor is synthesized in inactive form with little affinity for the operator 2. As trp accumulates, trp (co-repressor) binds to the repressors at its allosteric site and changes the repressor to active form 3. The activated repressor binds to operator which prevents RNA polymerase from binding to the promoter causing the operon to be switched off Lac operon

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Example of inducible operon Uses both negative and positive control Has a promoter which is recognized by the RNA polymerase which then initiates transcription. Has an operator which is a segment of DNA that a repressor binds to. The repressor when bound will physically obstruct the RNA polymerase from transcribing the genes Structural genes (in order) Lac Z: encodes b-galactosidase whose primary role is to convert disaccharide lactose to monosaccharide glucose and galactose Lac Y: primary structure of permease which facilitates the entry of lactose into the bacterial cell Lac A: codes for enzyme transacetylase involved in removal of toxic byproducts All three genes are closely linked Z-Y-A They are transcribed in as a single unit which forms polycistronic mRNA Details 1. Regulatory gene lacl is located outside the operon and codes for an allosteric repressor protein that can switch off the lac operon by binding to the operator 2. Lacl is synthesized in the active form and binds to the operator, repressing the operon

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3. When lactose is present, allolactose is formed by enzyme b-galactosidase (*note that even in the absence of lactose, the repressed operon still products low levels of b-galactosidase known as basal levels of expression. When it is induced, the level of gene expression would be much higher) 4. Allolactose acts as inducer which binds to the repressor and inactivates it by causing the repressor to undergo a conformation change in shape Lac operon Repressor gene lacl is constitutively transcribed in active form which binds to the operator and repress the operon By binding to the operator, RNA polymerase is unable to bind to the promoter and thus lac operon is switched off Only basal level of gene expression occurs Lactose enter the cell with help from premease Lactose converted to allolactose by b-galactosidase Allolactose binds allosterically to the lacl repressor and deactivates it Lacl repressor cannot bind to operator RNA polyermase can successfully bind to promoter and transcribe operon - Glucose is preferred over lactose - Since glucose is present, the bacterial cell will choose to save energy and resources by using up all glucose first (this type of control is called catabolite repression) - RNA polymerase have low affinity for the promoter even though repressor lacl does not bind to operator At low glucose level, the amount of camp receptor protein (CAP/CRP) with a small organic molecule cyclic AMP (cAMP) increase The high level of cAMP binds to allosteric site on CRP and the protein assumes it active shape binding to a specific site at the upstream end of the lac promoter The attachment of CRP enables RNA polymerase to bind more efficiently and start transcription (assuming that there is lactose present and the lacl repressor is not attached to the operator)

Conditions Absence of lactose

Presence of lactose

Presence of lactose and glucose

Presence of lactose and absence of glucose

At high glucose level, the cell does not want to activate the lac operon - cAMP decrease, ie low cAMP bind to CRP. CRP not active and (positive control of lac cannot engage operon. RNA polymerase binds weakly to the operon activated) operon and transcription is slow (assuming that there is lactose present and lacl repressor is inactive as allolactose is bounded to it) o Therefore, lac operons negative control is like an on-off switch while the positive control is like a volume switch Describe the concept of simple operon (using lac as example) o Genes which encode proteins involved in similar processors or pathways tend to be clustered and under coordinated control referred to as operon o A single on-off switch controls the transcription of the whole cluster of functionally related genes 9|Page Created by SefLRho (2012) DHS

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Operon may be defined as a unit of bacterial gene expression and regulation which includes structural genes, an operators and a promoter The operator serves as the on-off switch. It controls the access of RNA polymerase to the structural genes Repressors can bind to it The operon codes for a polycistronic mRNA Operon may be under positive or negative control Positive control: operon will only be expressed in the presence of the regulatory protein, activators Negative control: operon will only be transcribed in the absence of the regulatory protein, repressor Some operons may be under both controls (not mutually exclusive)

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